Mesenteric Artery Endothelial Cells Research Articles

TRPV4, TRPC1, and TRPP2 assemble to form a flow-sensitive heteromeric channel.

Transient receptor potential (TRP) channels, a superfamily of ion channels, can be divided into 7 subfamilies, including TRPV, TRPC, TRPP, and 4 others. Functional TRP channels are tetrameric complexes consisting of 4 pore-forming subunits. The purpose of this study was to explore the heteromerization of TRP subunits crossing different TRP subfamilies. Two-step coimmunoprecipitation (co-IP) and fluorescence resonance energy transfer (FRET) were used to determine the interaction of the different TRP subunits. Patch-clamp and cytosolic Ca(2+) measurements were used to determine the functional role of the ion channels in flow conditions. The analysis demonstrated the formation of a heteromeric TRPV4-C1-P2 complex in primary cultured rat mesenteric artery endothelial cells (MAECs) and HEK293 cells that were cotransfected with TRPV4, TRPC1, and TRPP2. In functional experiments, pore-dead mutants for each of these 3 TRP isoforms nearly abolished the flow-induced cation currents and Ca(2+) increase, suggesting that all 3 TRPs contribute to the ion permeation pore of the channels. We identified the first heteromeric TRP channels composed of subunits from 3 different TRP subfamilies. Functionally, this heteromeric TRPV4-C1-P2 channel mediates the flow-induced Ca(2+) increase in native vascular endothelial cells.

Inhibitory effect of 14,15-EET on endothelial senescence through activation of mTOR complex 2/Akt signaling pathways

Therapies to reverse the vascular endothelial aging process may play as a novel strategy for the treatment of cardiovascular diseases. 14,15-epoxyeicosatrienoic acid (14,15-EET) is a predominant cytochrome P450 epoxygenases-derived arachidonic acid metabolite and possesses multiple biological effects on the vascular system. The present study sought to investigate the roles of mammalian target of rapamycin complex 2 (mTORC2)/Akt signaling pathways in mediating the effect of 14,15-EET on endothelial senescence. By measuring the isometric tension in rat mesenteric arteries, we demonstrated that 14,15-EET improved the impaired endothelium-dependent vasodilatation in aged rats through activating mTORC2/Akt signaling pathway. Meanwhile, by promoting the formation of mTORC2 and the phosphorylation of Akt (Ser473), 14,15-EET inhibited the senescence of rat mesenteric arterial endothelial cells, which was not influenced by rapamycin but was significantly attenuated by Akt1/2 kinase inhibitor. The knockdown of Rictor gene by RNA interference abolished the inhibitory effect of 14,15-EET on endothelial senescence. Furthermore, 14,15-EET down-regulated the expression of p53 protein in aged endothelial cells. Meanwhile, the nuclear translocation of telomerase reverse transcriptase and the nuclear telomerase activity were also enhanced by treatment with 14,15-EET. Therefore, our present study suggests the crucial role of mTORC2/Akt signaling pathways in the inhibitory effects of 14,15-EET on the endothelial senescence. Our findings reveal important mechanistic clues to understanding of the effects of 14,15-EET on the endothelial functions.

Functional Role of TRPV4-K Ca 2.3 Signaling in Vascular Endothelial Cells in Normal and Streptozotocin-Induced Diabetic Rats

The small conductance and intermediate conductance Ca(2+)-activated K(+) channels are known to be involved in the endothelium-dependent hyperpolarization. Ca(2+) entry into endothelial cells stimulates these channels, causing membrane hyperpolarization in endothelial cells and underlying smooth muscle cells. In the present study, with the use of coimmunoprecipitation and double immunolabeling methods, we demonstrated a physical interaction of transient receptor potential vanilloid 4 (TRPV4) with K(Ca)2.3 in rat mesenteric artery endothelial cells. Acetylcholine and 4α-PDD mainly acted through TRPV4-K(Ca)2.3 pathway to induce smooth muscle hyperpolarization and vascular relaxation. K(Ca)3.1 was also involved in the process but at a much lesser degree than that of K(Ca)2.3. Stimulating TRPV4-K(Ca)2.3 signaling pathway also increased local blood flow in mesenteric beds and reduced systemic blood pressure in anesthetized rats. In streptozotocin-induced diabetic rats, the expression levels of TRPV4 and K(Ca)2.3 were reduced, which could be an underlying reason for the dysfunction of endothelium-dependent hyperpolarization in these animals. These results demonstrated an important physiological and pathological role of TRPV4-K(Ca)2.3 signaling pathway in vascular endothelial cells.

Transient receptor potential canonical type 3 channels facilitate endothelium-derived hyperpolarization-mediated resistance artery vasodilator activity

Microdomain signalling mechanisms underlie key aspects of artery function and the modulation of intracellular calcium, with transient receptor potential (TRP) channels playing an integral role. This study determines the distribution and role of TRP canonical type 3 (C3) channels in the control of endothelium-derived hyperpolarization (EDH)-mediated vasodilator tone in rat mesenteric artery. TRPC3 antibody specificity was verified using rat tissue, human embryonic kidney (HEK)-293 cells stably transfected with mouse TRPC3 cDNA, and TRPC3 knock-out (KO) mouse tissue using western blotting and confocal and ultrastructural immunohistochemistry. TRPC3-Pyr3 (ethyl-1-(4-(2,3,3-trichloroacrylamide)phenyl)-5-(trifluoromethyl)-1H-pyrazole-4-carboxylate) specificity was verified using patch clamp of mouse mesenteric artery endothelial and TRPC3-transfected HEK cells, and TRPC3 KO and wild-type mouse aortic endothelial cell calcium imaging and mesenteric artery pressure myography. TRPC3 distribution, expression, and role in EDH-mediated function were examined in rat mesenteric artery using immunohistochemistry and western blotting, and pressure myography and endothelial cell membrane potential recordings. In rat mesenteric artery, TRPC3 was diffusely distributed in the endothelium, with approximately five-fold higher expression at potential myoendothelial microdomain contact sites, and immunoelectron microscopy confirmed TRPC3 at these sites. Western blotting and endothelial damage confirmed primary endothelial TRPC3 expression. In rat mesenteric artery endothelial cells, Pyr3 inhibited hyperpolarization generation, and with individual SK(Ca) (apamin) or IK(Ca) (TRAM-34) block, Pyr3 abolished the residual respective IK(Ca)- and SK(Ca)-dependent EDH-mediated vasodilation. The spatial localization of TRPC3 and associated channels, receptors, and calcium stores are integral for myoendothelial microdomain function. TRPC3 facilitates endothelial SK(Ca) and IK(Ca) activation, as key components of EDH-mediated vasodilator activity and for regulating mesenteric artery tone.

Apigenin, a plant‐derived flavone, activates transient receptor potential vanilloid 4 cation channel

Transient receptor potential vanilloid 4 (TRPV4) is a Ca(2+) -permeable channel with multiple modes of activation. Apigenin is a plant-derived flavone, which has potential preventive effects on the development of cardiovascular disease. We set out to explore the effects of apigenin on TRPV4 channel activity and its role in vasodilatation. The effects of apigenin (0.01-30 µM) on TPRV4 channels were investigated in HEK293 cells over-expressing TRPV4, rat primary cultured mesenteric artery endothelial cells (MAECs) and isolated small mesenteric arterial segments using whole-cell patch clamp, fluorescent Ca(2+) imaging, intracellular recording and pressure myography. Whole-cell patch clamp and fluorescent Ca(2+) imaging in HEK cells over-expressing TRPV4 showed that apigenin concentration-dependently stimulated the TRPV4-mediated cation current and Ca(2+) influx. In MAECs, apigenin stimulated Ca(2+) influx in a concentration-dependent manner. These increases in cation current and Ca(2+) influx were markedly inhibited by TRPV4-specific blockers and siRNAs. Furthermore, pressure myography and intracellular recording in small third-order mesenteric arteries showed that apigenin dose-dependently evoked smooth muscle cell membrane hyperpolarization and subsequent vascular dilatation, which were significantly inhibited by TRPV4-specific blockers. TRPV4 blocker or charybdotoxin (200 nM) plus apamin (100 nM) diminished the apigenin-induced dilatation. This is the first study to demonstrate the selective stimulation of TRPV4 by apigenin. Apigenin was found to activate TRPV4 channels in a dose-dependent manner in HEK cells over-expressing TRPV4 and in native endothelial cells. In rat small mesenteric arteries, apigenin acts on TRPV4 in endothelial cells to induce EDHF-mediated vascular dilatation.

CaMKII regulates intracellular calcium stores of native endothelial cells from mesenteric arteries

Ca2+ homeostasis is crucial for endothelial function and the regulation of vascular tone. The Ca2+‐dependent kinase CaMKII is activated by Ca2+ pulsars and might play an important role in endothelial Ca2+ homeostasis. Thus, the involvement of endothelial CaMKII in the regulation of intracellular Ca2+ dynamics has been investigated in native endothelial cells of mesenteric arteries. Acute exposure to KN‐93 (10 μM), a CaMKII inhibitor, has little if any effect on Ca2+ pulsars frequency or amplitude. However, CaMKII inhibition induces pulsar sites recruitment as more pulsars sites are active in the presence of KN‐93 compared to control (25%). Interestingly, in absence of extracellular Ca2+, KN‐93 appears to empty intracellular Ca2+ stores. Indeed, exposure to ionomycin, to empty endoplasmic reticulum in absence of extrallular Ca2+, provoked a large and transient intracellular Ca2+ increase. However, preincubation with KN‐93 abolished the ionomycin‐induced Ca2+ transient. These results suggest that CaMKII might inhibit IP3R receptors and/or phosphorylates phospholamban to regulate ER Ca2+ content. Confocal microscopy revealed that CaMKIIα & β are colocalized with IP3R1 in native endothelium. Our data suggest that CaMKII is an important regulator of endothelial Ca2+ homeostasis and may represent a novel target for the treatment of pathologies like hypertension.Supported by FRSQ, CFI, CIHR and HSFC.

Total flavonoids of Flos Chrysanthemi protect arterial endothelial cells against oxidative stress

Ethnopharmacological relevanceTotal flavonoids of Flos Chrysanthemi (TFFC) are known to modulate vascular functions, but their effect on endothelial cells injured by oxidative stress is unknown. Our objective was to investigate the vasoprotective effect and mechanism of action of TFFC on rat mesenteric artery exposed to superoxide anions produced by pyrogallol. Materials and methodsThe vasoprotective effect and mechanism of action of TFFC on primary cultured rat mesenteric arterial endothelial cells and small mesenteric arteries was investigated using small-vessel myography, fluorescent Ca2+ measurement, fluorescent membrane potential measurement and oxidative fluorescent studies. ResultsExperiments using small-vessel myography of third-order rat mesenteric arterial rings showed that pretreatment with pyrogallol (10–1000μM), an auto-oxidizing source of superoxide anions, dose-dependently decreased ACh-induced endothelium-dependent relaxation. TFFC (2.5–320mg/L) evoked a concentration-dependent dilation (pD2: 29.6±0.276mg/L), which was weakened by ChTX plus apamin. TFFC markedly attenuated the inhibition of vasorelaxation induced by pyrogallol (Emax elevated from 50.4±7.36% to 86.2±3.61%, and pD2 increased from 6.74±0.06 to 7.28±0.12). Furthermore, in primary cultured endothelial cells, fluorescent Ca2+ measurement, fluorescent membrane potential measurement and oxidative fluorescent studies demonstrated that ACh-induced endothelial Ca2+ influx and hyperpolarization were significantly weakened by the increased basal superoxide level induced by pyrogallol. When the endothelial cells were concurrently exposed to TFFC, the impairment effect of oxidative stress on ACh-induced Ca2+ influx, hyperpolarization and vasorelaxation were attenuated due to its superoxide-lowering activity. ConclusionThis study shows that oxidative stress has a pronounced deleterious effect on EDHF-mediated vasorelaxation to ACh in rat mesenteric artery. TFFC has vasodilating effect and protects EDHF-mediated vasodilator reactivity from oxidative stress. Thus, our experiments suggest that TFFC is potentially useful for the development of therapeutic treatments for cardiovascular diseases associated with oxidative stress.

Disruption of Na + ,HCO 3 − Cotransporter NBCn1 (slc4a7) Inhibits NO-Mediated Vasorelaxation, Smooth Muscle Ca 2+ Sensitivity, and Hypertension Development in Mice

Background— Disturbances in pH affect artery function, but the mechanistic background remains controversial. We investigated whether Na + ,HCO 3 − cotransporter NBCn1, by regulating intracellular pH (pH i ), influences artery function and blood pressure regulation. Methods and Results— Knockout of NBCn1 in mice eliminated Na + ,HCO 3 − cotransport and caused a lower steady-state pH i in mesenteric artery smooth muscle and endothelial cells in situ evaluated by fluorescence microscopy. Using myography, arteries from NBCn1 knockout mice showed reduced acetylcholine-induced NO-mediated relaxations and lower rho-kinase-dependent norepinephrine-stimulated smooth muscle Ca 2+ sensitivity. Acetylcholine-stimulated NO levels (electrode measurements) and N-nitro-l-arginine methyl ester–sensitive l-arginine conversion (radioisotope measurements) were reduced in arteries from NBCn1 knockout mice, whereas relaxation to NO-donor S-nitroso-N-acetylpenicillamine, acetylcholine-induced endothelial Ca 2+ responses (fluorescence microscopy), and total and Ser-1177 phosphorylated endothelial NO-synthase expression (Western blot analyses) were unaffected. Reduced NO-mediated relaxations in arteries from NBCn1 knockout mice were not rescued by superoxide scavenging. Phosphorylation of myosin phosphatase targeting subunit at Thr-850 was reduced in arteries from NBCn1 knockout mice. Evaluated by an in vitro assay, rho-kinase activity was reduced at low pH. Without CO 2 /HCO 3 − , no differences in pH i , contraction or relaxation were observed between arteries from NBCn1 knockout and wild-type mice. Based on radiotelemetry and tail-cuff measurements, NBCn1 knockout mice were mildly hypertensive at rest, displayed attenuated blood pressure responses to NO-synthase and rho-kinase inhibition and were resistant to developing hypertension during angiotensin-II infusion. Conclusions— Intracellular acidification of smooth muscle and endothelial cells after knockout of NBCn1 inhibits NO-mediated and rho-kinase–dependent signaling in isolated arteries and perturbs blood pressure regulation.

Effect of Hydrogen Peroxide and Superoxide Anions on Cytosolic Ca2+: Comparison of Endothelial Cells from Large-Sized and Small-Sized Arteries

We compared the Ca2+ responses to reactive oxygen species (ROS) between mouse endothelial cells derived from large-sized arteries, aortas (aortic ECs), and small-sized arteries, mesenteric arteries (MAECs). Application of hydrogen peroxide (H2O2) caused an increase in cytosolic Ca2+ levels ([Ca2+]i) in both cell types. The [Ca2+]i rises diminished in the presence of U73122, a phospholipase C inhibitor, or Xestospongin C (XeC), an inhibitor for inositol-1,4,5-trisphosphate (IP3) receptors. Removal of Ca2+ from the bath also decreased the [Ca2+]i rises in response to H2O2. In addition, treatment of endothelial cells with H2O2 reduced the [Ca2+]i responses to subsequent challenge of ATP. The decreased [Ca2+]i responses to ATP were resulted from a pre-depletion of intracellular Ca2+ stores by H2O2. Interestingly, we also found that Ca2+ store depletion was more sensitive to H2O2 treatment in endothelial cells of mesenteric arteries than those of aortas. Hypoxanthine-xanthine oxidase (HX-XO) was also found to induce [Ca2+]i rises in both types of endothelial cells, the effect of which was mediated by superoxide anions and H2O2 but not by hydroxyl radical. H2O2 contribution in HX-XO-induced [Ca2+]i rises were more significant in endothelial cells from mesenteric arteries than those from aortas. In summary, H2O2 could induce store Ca2+ release via phospholipase C-IP3 pathway in endothelial cells. Resultant emptying of intracellular Ca2+ stores contributed to the reduced [Ca2+]i responses to subsequent ATP challenge. The [Ca2+]i responses were more sensitive to H2O2 in endothelial cells of small-sized arteries than those of large-sized arteries.

Opening of Small and Intermediate Calcium-Activated Potassium Channels Induces Relaxation Mainly Mediated by Nitric-Oxide Release in Large Arteries and Endothelium-Derived Hyperpolarizing Factor in Small Arteries from Rat

This study was designed to investigate whether calcium-activated potassium channels of small (SK(Ca) or K(Ca)2) and intermediate (IK(Ca) or K(Ca)3.1) conductance activated by 6,7-dichloro-1H-indole-2,3-dione 3-oxime (NS309) are involved in both nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-type relaxation in large and small rat mesenteric arteries. Segments of rat superior and small mesenteric arteries were mounted in myographs for functional studies. NO was recorded using NO microsensors. SK(Ca) and IK(Ca) channel currents and mRNA expression were investigated in human umbilical vein endothelial cells (HUVECs), and calcium concentrations were investigated in both HUVECs and mesenteric arterial endothelial cells. In both superior (∼1093 μm) and small mesenteric (∼300 μm) arteries, NS309 evoked endothelium- and concentration-dependent relaxations. In superior mesenteric arteries, NS309 relaxations and NO release were inhibited by both N(G),N(G)-asymmetric dimethyl-l-arginine (ADMA) (300 μM), an inhibitor of NO synthase, and apamin (0.5 μM) plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34) (1 μM), blockers of SK(Ca) and IK(Ca) channels, respectively. In small mesenteric arteries, NS309 relaxations were reduced slightly by ADMA, whereas apamin plus an IK(Ca) channel blocker almost abolished relaxation. Iberiotoxin did not change NS309 relaxation. HUVECs expressed mRNA for SK(Ca) and IK(Ca) channels, and NS309 induced increases in calcium, outward current, and NO release that were blocked by apamin and TRAM-34 or charybdotoxin. These findings suggest that opening of SK(Ca) and IK(Ca) channels leads to endothelium-dependent relaxation that is mediated mainly by NO in large mesenteric arteries and by EDHF-type relaxation in small mesenteric arteries. NS309-induced calcium influx appears to contribute to the formation of NO.

Exercise improves the dilatation function of mesenteric arteries in postmyocardial infarction rats via a PI3K/Akt/eNOS pathway-mediated mechanism

Myocardial infarction (MI) has been shown to induce endothelial dysfunction in peripheral resistance arteries and thus increase peripheral resistance. This study was designed to investigate the underlying mechanisms of post-MI-related dysfunctional dilatation of peripheral resistance arteries and, furthermore, to examine whether exercise may restore dysfunctional dilatation of peripheral resistance arteries. Adult male Sprague-Dawley rats were divided into three groups: sham-operated, MI, and MI + exercise. Ultrastructure and relaxation function of the mesenteric arteries, as well as phosphatidylinositol-3 kinase (PI3K), Akt kinases (Akt), endothelial nitric oxide synthase (eNOS) activity, and phosphorylation of PI3K, Akt, and eNOS by ACh were determined. Post-MI rats exhibited pronounced ultrastructural changes in mesenteric artery endothelial cells and endothelial dysfunction. In addition, the activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh were significantly attenuated in mesenteric arteries (P < 0.05-0.01). After 8 wk of exercise, not only did endothelial cells appeared more normal in structure, but also ameliorated post-MI-associated mesenteric arterial dysfunction, which were accompanied by elevated activities of PI3K, Akt, and eNOS, and their phosphorylation by ACh (P < 0.05-0.01). Importantly, inhibition of either PI3K or eNOS attenuated exercise-induced restoration of the dilatation function and blocked PI3K, Akt, and eNOS phosphorylation by ACh in the mesenteric arteries. These data demonstrate that MI induces dysfunctional dilation of peripheral resistance arteries by degradation of endothelial structural integrity and attenuating PI3K-Akt-eNOS signaling. Exercise may restore dilatation function of peripheral resistance arteries by protecting endothelial structural integrity and increasing PI3K-Akt-eNOS signaling cascades.

Steady state decrease of smooth muscle cell and endothelial cell pH prevents hypertension induced by angiotensin II or NO‐synthase inhibition

The role of steady state changes in smooth muscle and endothelial cell pH for vascular function is not known. In this study we knocked out the Na, HCO3‐cotransporter NBCn1 (slc4a7) in mice which caused a reduced pH in mesenteric artery smooth muscle and endothelial cells. Isolated arteries had reduced acetylcholine induced, NO‐mediated relaxations and rho‐kinase dependent smooth muscle Ca‐sensitivity. NBCn1 knockout mice were hypertensive, displayed attenuated blood pressure increase to NO‐synthase inhibition and blood pressure decrease to rho‐kinase inhibition and were resistant to developing hypertension during angiotensin II infusion. The increase of extracellular NO in the presence of acetylcholine was reduced in arteries from NBCn1 knockout mice while dilation to the NO‐donor S‐nitroso‐N‐acetylpenicillamine, acetylcholine‐induced endothelial Ca‐increase and endothelial NO‐synthase expression were unaffected. Rho‐kinase activity was reduced at low pH. In the absence of CO2/HCO3−, no differences were observed between isolated arteries from NBCn1 knockout and wild type mice. Using a knockout mouse to induce sustained intracellular acidification, we show a novel interaction between smooth muscle cell and endothelial cell steady state intracellular pH, vascular function and blood pressure regulation.

25-Hydroxycholesterol exerts both a cox-2-dependent transient proliferative effect and cox-2-independent cytotoxic effect on bovine endothelial cells in a time- and cell-type-dependent manner

Background25-hydroxycholesterol (25-OHC) is a product of oxidation of dietary cholesterol present in human plasma. 25-OHC and other oxidized forms of cholesterol are implicated in modulating inflammatory responses involved in development of atherosclerosis and colon carcinogenesis.MethodsPrimary lymphatic, venous and arterial endothelial cells isolated from bovine mesentery (bmLEC, bmVEC, bmAEC) were treated with 25-OHC and tested for several different cellular parameters.ResultsWe found 25-OHC to be a potent inducer of cyclooxygenase-2 (Cox-2, prostaglandin G-H synthase-2) expression in bovine mesenteric lymphatic, venous, and arterial endothelial cells. The induction of Cox-2 expression in endothelial cells by 25-OHC led to an initial increase in cellular proliferation that was inhibited by the Cox-2 selective inhibitor celecoxib (Celebrex). Prolonged exposure to 25-OHC was cytotoxic. Furthermore, endothelial cells induced to express Cox-2 by 25-OHC were more sensitive to the effects of the Cox-2 selective inhibitor celecoxib (Celebrex). These results suggest that some effects of 25-OHC on cells may be dependent on Cox-2 enzymatic activity.ConclusionsCox-2 dependent elevating effects of 25-OHC on endothelial cell proliferation was transient. Prolonged exposure to 25-OHC caused cell death and enhanced celecoxib-induced cell death in a cell-type dependent manner. The lack of uniform response by the three endothelial cell types examined suggests that our model system of primary cultures of bmLECs, bmVECs, and bmAECs may aid the evaluation of celecoxib in inhibiting proliferation of different types of tumour-associated endothelial cells.

Endothelial Epithelial Sodium Channel Inhibition Activates Endothelial Nitric Oxide Synthase via Phosphoinositide 3-Kinase/Akt in Small-Diameter Mesenteric Arteries

Recent studies have shown that the epithelial sodium channel (ENaC) is expressed in vascular tissue. However, the role that ENaC may play in the responses to vasoconstrictors and NO production has yet to be addressed. In this study, the contractile responses of perfused pressurized small-diameter rat mesenteric arteries to phenylephrine and serotonin were reduced by ENaC blockade with amiloride (75.1+/-3.2% and 16.9+/-2.3% of control values, respectively; P<0.01) that was dose dependent (EC(50)=88.9+/-1.6 nmol/L). Incubation with benzamil, another ENaC blocker, had similar effects. alpha, beta, and gamma ENaC were identified in small-diameter rat mesenteric arteries using RT-PCR and Western blot with specific antibodies. In situ hybridization and immunohistochemistry localized ENaC expression to the tunica media and endothelium of small-diameter rat mesenteric arteries. Patch-clamp experiments demonstrated that primary cultures of mesenteric artery endothelial cells expressed amiloride-sensitive sodium currents. Mechanical ablation of the endothelium or inhibition of eNOS with N(omega)-nitro-L-arginine inhibited the reduction in contractility caused by ENaC blockers. ENaC inhibitors increased eNOS phosphorylation (Ser 1177) and Akt phosphorylation (Ser 473). The presence of the phosphoinositide 3-kinase inhibitor LY294002 blunted Akt phosphorylation and eNOS phosphorylation and the decrease in the response to phenylephrine caused by blockers of ENaC, indicating that the phosphoinositide 3-kinase/Akt pathway was activated after ENaC inhibition. Finally, we observed that the effects of blockers of ENaC were flow dependent and that the vasodilatory response to shear stress was enhanced by ENaC blockade. Our results identify a previously unappreciated role for ENaC as a negative modulator of eNOS and NO production in resistance arteries.

Evidence for the presence of GPRC6A receptors in rat mesenteric arteries

In this study, the presence of GPRC6A receptors in rat mesenteric artery was investigated. In artery homogenates, GPRC6A mRNA was detected and Western blotting showed the presence of GPRC6A protein. Immunohistochemical studies revealed GPRC6A in both endothelial cells and myocytes. In whole vessel segments, the GPRC6A activators, 300 microM l-ornithine and 100 microM Al(3+), induced endothelium-dependent myocyte hyperpolarizations sensitive to 10 microM TRAM-34, a blocker of intermediate conductance, Ca(2+)-sensitive K(+) channels (IK(Ca)). Activation of IK(Ca) with calindol (300 nM; a positive allosteric Ca(2+)-sensing receptor - CaR - modulator) was inhibited by 500 nM ouabain (inhibition of rat type 2 and type 3 Na(+)/K(+)-ATPases) but unaffected by 30 microM Ba(2+) (blockade of inwardly rectifying K(+) channels). Neither l-ornithine nor Al(3+) activated CaRs heterologously expressed in CHO or HEK293 cells. In the presence of 300 microM l-ornithine or 100 microM Al(3+), myocyte hyperpolarizations to calindol were potentiated whereas this potentiation and hyperpolarizations to l-ornithine were lost following incubation with an anti-GPRC6A antibody. It is concluded that GPRC6A receptors are present on mesenteric artery endothelial cells and myocytes and that their activation selectively opens IK(Ca) channels. This triggers a ouabain-sensitive myocyte hyperpolarization suggesting a close functional relationship between GPRC6A, the IK(Ca) channel and type 2 and/or type 3 Na(+)/K(+)-ATPases.

Purinergic 2X1Receptors Mediate Endothelial Dependent Vasodilation to ATP

ATP is an important endogenous mediator in the cardiovascular system. It induces endothelium dependent vasodilation, but the precise receptor pathway activated in this response is currently under debate. We have used traditional bioassay techniques to show that ATP-induced vasodilation in mesenteric vessels is endothelium-dependent. Furthermore, ATP-induced vasodilation was inhibited by both suramin and 2',3'-O-(2,4,6-trinitrophenyl)-ATP (TNP-ATP), consistent with a P2X(1)-, P2X(2)-, or P2X(3)-mediated event and was not potentiated by ivermectin, indicating that these responses were not P2X(4) receptor-mediated. ATP did not induce vasodilation in vessels from P2X (-/-)(1) mice, confirming an absolute requirement for this receptor. Finally, in pure cell populations of mouse mesenteric artery endothelial cells, we show that P2X(1) mRNA is specifically expressed. However, in line with observations in the brain, the P2X(1) present in endothelial cells does not seem to be recognized by conventional antibodies. Together, these results show that ATP-induced vasodilation is mediated by P2X(1) receptor activation on mesenteric arterial endothelial cells. These observations establish a critical role for P2X(1) receptors in the ATP vasodilator pathway.

Regional Differences in the Regulation of Vascular Smooth Muscle Relaxation by the Endothelium in Health and in Diabetes

1 Monash University, Melbourne, Australia. Correspondence should be addressed to M. Tare (e-mail: marianne.tare@med.monash.edu.au). The potent vasodilator influence of the vascular endothelium is compromised in diabetes, and this contributes to the increased vascular tone and reduced tissue perfusion characteristics of this disease. Regional differences exist in the extent of this vasodilator dysfunction, and in larger arteries the focus has been on nitric oxide (NO). In smaller arteries and arterioles, it is emerging that the actions of endothelium-derived hyperpolarizing factor (EDHF) are diminished in diabetes. In the mesenteric arteries of streptozotocin (STZ)-induced diabetic rats, smooth muscle (SM) hyperpolarization and relaxation attributed to EDHF is halved. It is unclear whether the dysfunction is due to changes in the generation of hyperpolarization in the endothelial cells (EC), or to spread of hyperpolarization to the SM via myoendothelial gap junctions (MEGJ), or in the SM response. In our study, we addressed this question. Responses were compared with those of the femoral artery where the entire endothelium-dependent relaxation is mediated by NO and is resistant to diabetes. Male Wistar rats were injected either with 60 mg/kg STZ dissolved in citrate buffer or with the buffer alone (control) into the tail vein, and 8 weeks after injection they were killed by exsanguination. Segments of the mesenteric and femoral arteries were isolated, secured to a recording chamber (or a myograph), and superfused with warmed oxygenated physiological saline. To isolate the EDHF response, 10 M N-nitro-L-arginine methyl ester and 10 M indomethacin were added to this saline. Intracellular glass microelectrodes with tips filled with 2% Lucifer Yellow CH were used to record the membrane potential of EC and SMC. Blood glucose levels were 6-fold higher in STZinjected rats. Acetylcholine evoked concentrationdependent hyperpolarization in the mesenteric artery EC and SMC of control rats. Hyperpolarization in EC was halved in diabetic rats, and this accounts for the reduced hyperpolarization and relaxation in SMC. Hyperpolarization, selectively evoked in the SMC by the ATP-sensitive potassium channel opener levcromakalim, spread to the EC via MEGJ, and this was not altered by diabetes. Femoral arteries lack MEGJ; the fact that the ability of EC to generate hyperpolarization is not influenced by diabetes is intriguing. Impaired generation of EC hyperpolarization accounts for the reduction in EDHF-mediated SMC hyperpolarization and relaxation in the mesenteric arteries in diabetes. The EC–SMC communication via MEGJ remains functional in diabetes. In contrast, EC hyperpolarization was preserved in the femoral arteries. Thus, there are regional differences in the impact of diabetes on EC hyperpolarization, and this may be related to the existence/absence of electrical and/or chemical contacts between EC and SMC.

Expression of Ryanodine Receptor 3 and Trp Channels in Endothelium of Human Mesenteric Artery: A Single-Cell Rt-Pcr and Patch-Clamp Analysis in Situ .

P146 Ca 2+ mobilization plays an important role in endothelial function by stimulating Ca 2+ -dependent synthesis of vasodilating factors. In addition to InsP 3 - mediated Ca 2+ store depletion, Ca 2+ release from ryanodine-sensitive pools and Ca 2+ -influx through cation channels of the TRP-gene family have been suggested to be involved in endothelial Ca 2+ signaling. In cultured endothelial cells (EC) the function and expression of ryanodine-receptors (RyR) and TRP channels might differ substantially from those in native endothelium of human blood vessels. We, therefore, characterized expression and function of RyR and TRP channels in EC of small human mesenteric artery (MA) by use of single-cell RT-PCR and patch-clamp techniques in situ. MA were isolated from colon specimens of patients subjected to hemicolectomy. For single-cell RT-PCR and PC experiments single human mesenteric artery EC (HMAEC) were harvested directly from the luminal vessel with the patch pipette. Expression of the RyR subtype 3, but not of the RyR1 and RyR2 subtpyes was detected in 25% of HMAEC samples. Correspondingly, in PC experiments in HMAEC, application of caffeine (0.5 mM) induced Ca 2+ -release from ryanodine-sensitive stores and subsequently activation of Ca 2+ -activated K + channels leading to a sustained endothelial hyperpolarization from a resting potential of 28 ± 3 mV to -46 ± 4 mV. Single HMAEC expressed the TRP subtypes, TRP1 and TRP3, but not TRP 4 and 6. The TRP1 was the predominantly expressed TRP subtype as expression was detected in 16% of HMAEC. TRP3 expression was detected in only 3% of HMAEC. In single-channel and whole-cell patch clamp experiments in HMAEC, InsP 3 -mediated Ca 2+ -store depletion in response to bradykinin (100 nM) activated TRP related nonselective cation currents leading to Ca 2+ entry. In conclusion, Ca 2+ release from ryanodine-sensitive stores mediated by RyR3 and Ca 2+ entry through TRP1 might represent important components of endothelial Ca 2+ signaling and thereby of endothelial function in intact human blood vessels.

P–174 - Comparative study of characteristics of rabbit aortic and mesenteric arterial endothelial cells

P–174 - Comparative study of characteristics of rabbit aortic and mesenteric arterial endothelial cells

Ultrastructural localization of nitric oxide synthase and endothelin in the renal and mesenteric arteries of the golden hamster: differences during and after arousal from hibernation.

This is a study of the electron-immunocytochemical localization of nitric oxide synthase (type III) and endothelin in renal and mesenteric artery endothelial cells of normal (active) and hibernating hamsters, as well as hamsters exposed to the cold but not hibernating, and hamsters aroused for 2h following hibernation. In the renal artery of hibernating hamsters and cold-exposed hamsters, a subpopulation of nitric oxide synthase-positive endothelial cells displayed immunoprecipitate predominantly in the vicinity of the Golgi complex indicating intracellular translocation from the cytoplasm to the Golgi complex. In hibernating animals, the percentages of both nitric oxide synthase-positive and endothelin-positive endothelial cells were notably lower than those observed either in active, cold-exposed or aroused animals. These changes may reflect a reduced endothelial contribution to the maintenance of vascular tone in these vessels during hibernation and an upregulation of expression of nitric oxide synthase and endothelin in the endothelium early on during arousal from hibernation.